DESCRIPTION (Applicant's Abstract): We plan to develop, test and use computational tools for a fine scale investigation of the evolutionary history of microbial genomes. Understanding the evolutionary dynamics underlying the emergence of a pathogen with novel disease phenotypes will be greatly advanced by comparisons of the complete genome contents, organization and gene expression patterns of closely related organisms. We have designed an efficient and effective whole-genome pair-wise alignment tool and applied it to comparisons of the genomes of distinct pathogenic and non-pathogenic lineages of E. coli to delineate the genetic elements that distinguish among these strains. This preliminary work generated numerous insights into evolution of the E. coli genome in general and the relative roles of horizontal transfer and clonal divergence. We now propose further testing on a more diverse selection of pathogenic and non-pathogenic microorganisms to investigate the generality of what we have learned about evolution of microbial genomes and the robustness of our method. We also propose to expand this method to construct multiple alignments of all homologous regions of three or more genomes and to use phylogenetic analysis to partition the genome-scale multiple alignment into evolutionarily homogeneous units. Finally, we plan to develop an interactive Web-base viewer to integrate the results of these analyses with genome annotations and gene expression patterns. A whole-genome comparative approach to molecular evolution is the essential companion for an organismal level approach to studying the evolution of disease.